CN104284987A - Method for detecting genes sensitive to low-level ionizing radiation, and gene detected by the method - Google Patents
Method for detecting genes sensitive to low-level ionizing radiation, and gene detected by the method Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The present invention relates to a method for detecting genes sensitive to low-level ionizing radiation and genes detected by the method, more specifically to a method for detecting genes, sensitive to low-level ionizing radiation and discovered in a carcinogenic entity and verified in a normal entity, by subjecting a cancerous AKR/J mouse and a normal ICR mouse to low-level radiation, collecting thymus therefrom, classifying glycometabolism-related genes via microarray processing of the thymus, and amplifying the genes and measuring the levels of gene expression. Thus, the present invention allows a gene having a specific reaction to radiation to be accurately detected by preventing the interference of confounding variables.
Description
Technical field
The detection method that the present invention relates to a kind of gene to low strength ionizing radiation sensitivity and the gene detected by described method, particularly relate to and a kind of mouse is brought out to cancer and normal mouse irradiates low strength radioactive rays, thus be sorted in by thymus gland normal mouse to bring out the gene to low strength ionizing radiation sensitivity of the relevant glycometabolic gene jointly observed specifically in mouse detection method with cancer.
Background technology
Along with the increase of utilization of industry and medical radioactive rays, the research about the impact on human body is diversely carried out, and mainly utilizes the perpetual object of the cancer therapy aspect of radiation exposure.Known high dosage ionizing radiation can cause DNA damage, gene to be out of shape and comprise the disease of cancer, however the radioactive rays of the dose rate of the dosage of below 200mGy and less than 6mGy/ hour but activate immunity react and suppress the appearance of cancer.
Usually, for radioactive rays and carcinogenic between the research of dependency (especially Gene response) carry out so far, but be mingled with the perturbing variables of the reliability hindering its result.But, for major part research so far, make a part of gene be out of shape or utilize JEG-3, the diversified reaction that therefore cell of formation health, tissue, internal organs cannot be described and observe in the health stage.That is, assess Gene response owing to utilizing common mouse, therefore expressing gene is varied, moreover, because the appearance of cancer is not confined to specific internal organs, therefore there is the problem being difficult to explain Gene response.
For utilize the method for cell in prior art in order to cancer research for, cancer cells gene being changed or p53 important in carcinogenic aspect for radiation exposure is lacked, therefore fundamentally different from Normocellular reaction, therefore there is the problem that its result cannot be applied to individuality, and in order to alleviate such shortcoming, utilize the mouse of gene similar to the mankind more than 95% and the carcinogenic research carried out for radioactive rays.But, because the natural canceration incidence of common mouse is very low, therefore utilize diversified cancer research model mice.
Existing research contents make use of diversified method in order to excavate to the gene be associated with carbohydrate metabolism of low strength (0.7mGy/ hour) radioactive rays sensitivity.But the gene be associated with carbohydrate metabolism disclosed in the present invention did not but disclose it in the prior art for the gene to low strength (0.7mGy/ hour) radioactive rays sensitivity.
In the excavation of the spectrum of the carbohydrate metabolism genes involved to low strength (0.7mGy/ hour) radioactive rays sensitivity of the present invention, there is following prior art.
1. cancer cells shows sugar absorption and activates feature (Warburg O, Science 1956 with sugar decomposition; 123:309-314).
2. the carbohydrate metabolism be activated suppresses p53 active in thymus gland, and suppresses puma to induce, and suppresses balance to have an impact to the necrocytosis of Bcl2family protein expression, and maintains existence (Zhao Y et.al., the J Biol Chem 2008 of cancer; 283:36344-36353).
3. the Akt1 of illuminated ionizing radiation expresses necrocytosis in the ileum of the mouse be suppressed increases (Plastaras etc., 2008).
Based on this, present inventor excavates out and composes the carbohydrate metabolism genes involved of low strength ionizing radiation sensitivity, thus completes the present invention.
Summary of the invention
Technical problem
The detection method that the object of the present invention is to provide a kind of gene to low strength ionizing radiation sensitivity and the gene detected by described method.
Technical scheme
In order to reach object as above, the invention provides a kind of detection method of the gene to low strength ionizing radiation sensitivity, comprising the steps: I) low strength radioactive rays are irradiated to the AKR/J mouse and normal ICR mouse of bringing out cancer; II) from described AKR/J mouse and ICR mouse, thymus gland is gathered; III) microarray is performed to described thymus gland; IV) classify about glycometabolic gene by described microarray; V) increase described gene, and measure expression amount, and, bring out individuality from cancer and excavate and verify in normal individual.
And, the invention provides a kind of radiation-sensitive or radioactive rays carcinogenic diagnosis mark, comprising: from by as the base sequence participating in the gene selected group that IRS1 (NM_010570), the Gl ut1 (MM_011400) of the relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form.
And, the invention provides a kind of radiation-sensitive or the carcinogenic diagnosis external member of radioactive rays that comprise mark as above.
Further, the invention provides a kind of radiation-sensitive or the carcinogenic diagnosis microarray of radioactive rays that comprise mark as above.
And, the invention provides a kind of detection method that can measure radiation-sensitive or the carcinogenic gene of radioactive rays, comprise the steps: I) radioactive rays are irradiated to the Mammals with thymic carcinoma except the mankind; II) thymic tissue extracted from the Mammals of illuminated described radioactive rays is contacted with Test Materials; III) measure from by as the expression change participating in the gene selected group that IRS1 (NM_010570), the Glut1 (MM_011400) of the relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form by described thymic tissue.
Below, the present invention is described in detail.
At radioactive rays in the affecting of human body, much carcinogenic research is carried out so far, but owing to make use of cancer cells, the cell strain of gene distortion or common mouse, is therefore difficult to diversified somatic reaction (Gene response) is described.Especially, on individual level, and the gene profile that with carbohydrate metabolism be associated responsive to ionizing radiation had not been excavated out and functions.To this, in the present invention, 1) for the AKR/J mouse that normal ICR mouse and thymic carcinoma fall ill, after low strength (0.7mGy/ hour) radiation exposure (carcinogenic stimulating factor), excavate out the spectrum of the carbohydrate metabolism genes involved to low strength radioactive rays sensitivity analytic function expressed specifically in thymus gland, then, 2) for using the integrant of the carbohydrate metabolism genes involved spectrum for can be used for diagnosing thymic carcinoma stage of growth.
The invention provides a kind of detection method of the gene to low strength ionizing radiation sensitivity, comprise the steps: I) low strength radioactive rays are irradiated to the AKR/J mouse and normal ICR mouse of bringing out cancer; II) from described AKR/J mouse and ICR mouse, thymus gland is gathered; III) microarray is performed to described thymus gland; IV) classify about glycometabolic gene by described microarray; V) increase described gene, and measure expression amount, and, bring out individuality from cancer and excavate and verify in normal individual.
In the detection method of the gene to low strength ionizing radiation sensitivity of the present invention, described low strength ionizing radiation preferably finally irradiates the γ line Cs-137 of the dose radiation of 1.7Gy with the intensity of 0.7mGy/ hour, and described method of the present invention is preferably used as the making of thymic carcinoma diagnosis external member, the canceration progress of cancer patients and the assessment for the treatment of degree, industry and medical treatment be engaged in personnel radiation exposure and carcinogenic between relevance evaluation, radioactive rays and carcinogenic cause-effect relationship are assessed, biological dose for radiation exposure is assessed, or based on the initiation of the thymic carcinoma of low strength radioactive rays and the index of progress degree assessment.
Further, in the detection method of the gene to low strength ionizing radiation sensitivity of the present invention, described cancer is preferably thymic carcinoma, at this, described Step II) in the collection of thymus gland preferably starting due to cancer to occur dead time point carries out.
And, in the detection method of the gene to low strength ionizing radiation sensitivity of the present invention, preferably, described relevant glycometabolic gene is from by IRS1 (NM_010570), Glut1 (MM_011400), Glut4 (NM_009204), the gene selected in the group that LPK (NM_013631) and G6pc (NM_008061) is formed, at this, preferably, described IRS1 (NM_010570) gene is by the primer amplification being designated as sequence number 1 and 2, Glut1 (MM_011400) gene is by the primer amplification being designated as sequence number 3 and 4, Glut4 (NM_009204) gene is by the primer amplification being designated as sequence number 5 and 6, LPK (NM_013631) gene is by the primer amplification being designated as sequence number 7 and 8, and G6pc (NM_008061) gene is by the primer amplification being designated as sequence number 9 and 10.
For described step IV) thus sayed by described microarray classification carbohydrate metabolism dependency basis, it is characterized in that, individuality is brought out compared to the cancer of irradiating before radioactive rays, more favor in bringing out from the cancer of irradiating after radioactive rays the gene being detected process LAN or low expression in individuality by microarray, then utilize sequence number to be that the primer of No. 1 ~ No. 10 is verified, and separate the original shape of the gene of bright described process LAN or low expression by the function retrieval of gene.Describe in detail about in the embodiment that microarray analysis will be described below, the function retrieval of gene is then implemented by biomolecule information database DAVID (http://apps1.niaid.nih.gov/david/) and Pubmed database (http://www.ncbi.nlm.nih.gov/) in embodiment described later, but is not limited thereto.
" gene to low strength ionizing radiation sensitivity " that use in the present invention refer to irradiate radioactive rays before cancer bring out individual Comparatively speaking at the gene irradiating the cancer after radioactive rays with bringing out distinctiveness in individuality process LAN or low expression.That is, representing and to bring out in individuality the gene of the change of abduction delivering pattern because radioactive rays stimulate in cancer, and can be the target gene (that is, tumour forms gene or tumor suppressor gene) joined with specific related to cancer.Detect such cancer specific gene, thus the molecular mechanism of establishing for the radiation cure of cancer patients, can contribute to the raising of radiation cure effect thus, moreover, regulate it to express by excavating new tumour and forming gene or tumor suppressor gene, thus can be that the exploitation of cancer therapeutic agent in molecular biology level or methods for the treatment of lay the foundation.
And, the invention provides a kind of radiation-sensitive or radioactive rays carcinogenic diagnosis mark, comprising: from by as the base sequence participating in the gene selected group that IRS1 (NM_010570), the Gl ut1 (MM_011400) of the relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form.
And, the invention provides a kind of radiation-sensitive or the carcinogenic diagnosis external member of radioactive rays that comprise mark as above.
Further, the invention provides a kind of radiation-sensitive or the carcinogenic diagnosis microarray of radioactive rays that comprise mark as above.
And, the invention provides a kind of detection method that can measure radiation-sensitive or the carcinogenic gene of radioactive rays, comprise the steps: I) radioactive rays are irradiated to the Mammals with thymic carcinoma except the mankind; II) thymic tissue extracted from the Mammals of illuminated described radioactive rays is contacted with Test Materials; III) measure from by as the expression change participating in the gene selected group that IRS1 (NM_010570), the Glut1 (MM_011400) of the relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form by described thymic tissue.
In the present invention, for the ICR mouse as the AKR/J mouse of thymic carcinoma research model and the mouse kind as health, irradiate low strength (0.7mGy/ hour) γ line Cs-137, and start to occur dead time point (100 days) acquires thymus gland because of thymic carcinoma AKR/J mouse.Microarray is performed for the thymus gland gathered, then David's (DAVID) biomolecule information database is utilized and the carbohydrate metabolism genes involved of reaction low strength (0.7mGy/ hour) radioactive rays being shown to sensitivity of classifying, and carbohydrate metabolism genes involved low strength (0.7mGy/ hour) radioactive rays being shown to sensitivity response is carried out nucleic acid amplification, and determine expression amount.
Its result, excavated out by the present invention and 5 important genes of carbohydrate metabolism (IRS1, Glut1, Glut4, LPK and G6pc) aspect of sensitivity response are shown to low strength (0.7mGy/ hour) radioactive rays, and disclose the function of carbohydrate metabolism (IRS1, Glut1, Glut4, LPK and G6pc) genes involved low strength (0.7mGy/ hour) radioactive rays being shown to sensitivity response easy-to-understandly.Further, regulation is observed dead the 100th day and gathers thymus gland because of thymic carcinoma, thus observes glucose metabolism genes reaction low strength radioactive rays being shown to reaction with can having consistency.
Therefore, the present invention can be applied as following purposes widely: 1) for developing the carbohydrate metabolism genes involved spectrum of thymic carcinoma diagnosis diagnostic kit; 2) the carcinogenic cause-effect relationship evaluation index of personnel is engaged in the industry of living in low strength radioactive rays environment and medical treatment; 3) for diagnosing cancer patient canceration and establish the information gene profile for the treatment of strategies; 4) can be used for distinguishing radiation exposure and thymic carcinoma occur between causal index; 5) the new gene index for assessing for the biological dose of low strength radiation exposure.In addition, 6) targeted therapy for low strength radiation exposure is applicable as to the carbohydrate metabolism signal transmission of ionizing radiation sensitivity.
Beneficial effect
For the detection method of the gene for low strength ionizing radiation sensitivity formed as described above, low strength (0.7mGy/ hour) the radiation-sensitive carbohydrate metabolism index of correlation gene profile for making thymic carcinoma diagnosis external member can be utilized as, and low strength (0.7mGy/ hour) the radiation-sensitive carbohydrate metabolism index of correlation gene of canceration progress and the treatment degree that can be used for assessment of cancer patient can be utilized as.And, can be utilized as can be used for assessing industry and medical treatment be engaged in personnel radiation exposure and carcinogenic between low strength (0.7mGy/ hour) the radiation-sensitive carbohydrate metabolism index of correlation gene of dependency, and can be utilized as can be used for assessing radioactive rays and carcinogenic between causal low strength (0.7mGy/ hour) radiation-sensitive carbohydrate metabolism index of correlation.And, the new index for assessing for the biological dose of radiation exposure can be utilized as, and can be utilized as assessment based on low strength (0.7mGy/ hour) radioactive rays thymic carcinoma occur and progress degree, inhibition low strength radiation-sensitive carbohydrate metabolism index of correlation gene.
Accompanying drawing explanation
Fig. 1 is the figure that easy-to-understand map solves carbohydrate metabolism genes involved function thymic carcinoma being inhibited due to low strength (0.7mGy/ hour) radiation exposure.
The variation diagram of Fig. 2 represents raises with after low strength (0.7mGy/ hour) radiation exposure AKR/J mouse and ICR mouse, and regulation AKR/J mouse starts because of thymic carcinoma occur dead time point (100 days) and gather thymus gland and measure thymic weight, and analyze as index the carbohydrate metabolism genes involved of radioactive rays sensitivity is reacted.
Embodiment
Below, the present invention is further illustrated by embodiment.But, following examples just in order to the present invention is described, interest field of the present invention be not limit by following examples.
< embodiment 1>
Female AKR/J mouse as thymic carcinoma research model and normal Female ICR mice is bought from Japanese SLC company.Utilize γ line generating unit (IBL 147C, CIS biological international (CIS bio internat ional), France) and to described AKR/J mouse irradiate (
137cs, 0.7mGy/ hour) low strength radioactive rays and make final dose reach 4.5Gy.The mouse of terminating low strength radiation exposure is moved on to the aseptic feeding facility shielding radioactive rays and raises 100 days and observe the generation of thymic carcinoma.In order to genetic analysis, low strength (0.7m Gy/ hour) radioactive rays are irradiated to the normal mouse (ICR mouse) of raising separately under identical experiment condition, and by thymus gland IQF in liquid nitrogen that 100 days gather later, then perform genetic analysis.
< embodiment 2> microarray and genetic analysis
The result of described embodiment 1, utilize cancer research model mice (AKR/J mouse) and the gene be associated with carbohydrate metabolism excavated out low strength (0.7mGy/ hour) radioactive rays sensitivity, and proved in normal mouse (ICR mouse).That is, from the thymus gland of the AKR/J mouse and ICR mouse of having irradiated low strength (0.7mGy/ hour) radioactive rays, excavate out the gene be associated with carbohydrate metabolism that low strength radioactive rays are induced reaction specifically and function is explained.Utilize David (DAVID) biomolecule information database, quantitative nucleic acid amplification method and statistics program SAS (ANOVA and t-test) and analyze.
In order to confirm its result, nucleic acid amplification is carried out to described gene.Specifically, in order to carry out microarray to the thymus gland gathered from the AKR/J mouse of having irradiated low strength (0.7mGy/ hour) radioactive rays and ICR mouse and be directed to relevant glycometabolic gene measurement expression amount low strength radioactive rays being shown to sensitivity response, utilize the primer that is recorded in table 1 below and increase.
[table 1]
Further, raise after low strength (0.7mGy/ hour) radioactive rays are irradiated to AKR/J mouse and ICR mouse, and start to occur dead time point (100 days) acquires thymus gland because of thymic carcinoma AKR/J mouse.By the thymus gland microarray gathered, and distinguish responsive reaction is shown and the gene be associated with carbohydrate metabolism to low strength radioactive rays, then amplification of nucleic acid and determine expression amount.Its result, in the mouse of irradiating low strength radioactive rays, relevant glycometabolic gene (IRS1, Glut1, Glut4, LPK and G6pc) is responsive to the reaction of low strength radioactive rays.Its result is recorded in table 2 below.
[table 2]
* multiple ± standard deviation is expressed
And easy-to-understand map solves the relevant glycometabolic gene function making thymic carcinoma be inhibited owing to irradiating low strength (0.7mGy/ hour) radioactive rays in FIG.The irradiation of low strength radioactive rays makes insulin signaling transmission (IRS1), sugar absorbs (Glut1 and Glut4), sugar decomposition (LPK) gene and new sugared expression of synthesizing (G6pc) gene and reduces, and inhibits thymic carcinoma.
And, in fig. 2, raise after low strength (0.7mGy/ hour) radioactive rays are irradiated to AKR/J mouse and ICR mouse, and regulation AKR/J mouse starts because of thymic carcinoma to occur dead time point (100 days), and gather thymus gland and measure thymic weight, and analyze the relevant glycometabolic Gene response to radioactive rays sensitivity as index.By the present invention, specify to start because of thymic carcinoma to occur dead pathogenesis of cancer initial stage, and gather thymus gland and compare weight, thus can consistency be remained and measure relevant glycometabolic gene low strength radioactive rays being shown to sensitivity response.
Claims (11)
1., to a detection method for the gene of low strength ionizing radiation sensitivity, comprise the steps:
I) low strength radioactive rays are irradiated to the AKR/J mouse and normal ICR mouse of bringing out cancer;
II) from described AKR/J mouse and ICR mouse, thymus gland is gathered;
III) microarray is performed to described thymus gland;
IV) classify about glycometabolic gene by described microarray;
V) increase described gene, and measure expression amount,
And, bring out individuality from cancer and excavate and verify in normal individual.
2. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 1, it is characterized in that, described low strength ionizing radiation is the γ line Cs-137 finally irradiating the dose radiation of 1.7Gy with the intensity of 0.7mGy/ hour.
3. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 1, it is characterized in that, described method be used as the making of thymic carcinoma diagnosis external member, the assessment of the canceration progress of cancer patients and treatment degree, industry and medical treatment be engaged in personnel radiation exposure and carcinogenic between relevance evaluation, radioactive rays and the carcinogenic cause-effect relationship index assessing, the biological dose of radiation exposure assessed or assesses based on initiation and the progress degree of the thymic carcinoma of low strength radioactive rays.
4. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 1, it is characterized in that, described cancer is thymic carcinoma.
5. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 1, is characterized in that, described Step II) in the collection of thymus gland be starting due to cancer to occur dead time point carries out.
6. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 1, it is characterized in that, described about glycometabolic gene be the gene selected from the group be made up of IRS1 (NM_010570), Glut1 (MM_011400), Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061).
7. the detection method to the gene of low strength ionizing radiation sensitivity as claimed in claim 6, it is characterized in that, described IRS1 (NM_010570) gene is by the primer amplification being designated as sequence number 1 and 2, Glut1 (MM_011400) gene is by the primer amplification being designated as sequence number 3 and 4, Glut4 (NM_009204) gene is by the primer amplification being designated as sequence number 5 and 6, LPK (NM_013631) gene is by the primer amplification being designated as sequence number 7 and 8, and G6pc (NM_008061) gene is by the primer amplification being designated as sequence number 9 and 10.
8. a radiation-sensitive or the carcinogenic diagnosis mark of radioactive rays, comprising:
From by as the base sequence participating in the gene selected group that IRS1 (NM_010570), the Glut1 (MM_011400) of relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form.
9. radiation-sensitive or the carcinogenic diagnosis external member of radioactive rays, comprise mark according to claim 8.
10. radiation-sensitive or the carcinogenic diagnosis microarray of radioactive rays, comprise mark according to claim 8.
11. 1 kinds, for measuring the detection method of radiation-sensitive or the carcinogenic gene of radioactive rays, comprise the steps:
I) radioactive rays are irradiated to the Mammals with thymic carcinoma except the mankind;
II) thymic tissue extracted from the Mammals of illuminated described radioactive rays is contacted with Test Materials;
III) measure from by as the expression change participating in the gene selected group that IRS1 (NM_010570), the Glut1 (MM_011400) of the relevant glycometabolic gene that thymic carcinoma worsens, Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061) form by described thymic tissue.
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| KR10-2012-0049544 | 2012-05-10 | ||
| PCT/KR2012/003928 WO2013168844A1 (en) | 2012-05-10 | 2012-05-18 | Method for detecting genes sensitive to low-level ionizing radiation, and gene detected by the method |
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| KR101714383B1 (en) * | 2014-10-22 | 2017-03-10 | 한국수력원자력 주식회사 | Detection method of sensitive genes for low level radioactiveray |
| KR101881874B1 (en) * | 2016-04-29 | 2018-07-26 | 한국수력원자력 주식회사 | Methods for Preventing the Malignization of Normal Cells by Low Dose Radiation |
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| KR101875116B1 (en) * | 2016-10-28 | 2018-07-06 | 한국수력원자력 주식회사 | Immune-response indicator protein of low-dose radiation exposure and detecting method thereof |
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| US9714453B2 (en) | 2017-07-25 |
| US20170058359A1 (en) | 2017-03-02 |
| US20170073773A1 (en) | 2017-03-16 |
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| US9708668B2 (en) | 2017-07-18 |
| JP2015515865A (en) | 2015-06-04 |
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| JP6064039B2 (en) | 2017-01-18 |
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